Chronic viral hepatitis and its long-term sequelae, cirrhosis and HCC, represent a major global health problem. Considerable progress has been made in the control and treatment of chronic viral hepatitis, but further progress will depend on a more thorough knowledge of the molecular mechanisms of pathogenesis. The Hepatic Pathogenesis Section (HPS) has developed a comprehensive research program to study the pathogenesis of acute and chronic liver diseases in humans in collaboration with the Liver Transplanation Center and the Liver Unit of the University of Cagliari, Italy, which is an invaluable source of clinical samples. Our main research strategy is to combine basic research with clinical medicine. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF) While the liver damage in classic acute hepatitis B is T-cell mediated, the pathogenesis of HBV-associated ALF is unknown. By gene expression analysis, we demonstrated that ALF is characterized by an overwhelming B-cell signature centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, and complement deposition. By phage-display libraries, we discovered that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg);that these antibodies display a restricted variable heavy chain (VH) repertoire and are in germline configuration;and that unrelated ALF patients use an identical unmutated VH gene (VH1-3) for both IgG and IgM anti-HBc antibodies. Thus, our data strongly suggest that HBV-associated ALF is mediated by a T cell-independent intrahepatic B-cell response against the core antigen of HBV that is associated with complement-mediated massive hepatocellular damage (Farci et al. PNAS 2010). In collaboration with Dr. Steven from the LSBR, NIAMS, we have characterized the epitope of these antibodies using cryo-electron microscopy, image reconstruction, and molecular modeling. The antibodies associated to ALF bind to a region in which epitopes have not been detected before. Thus, our data are consistent with the hypothesis that antibodies directed to a particular epitope of HBcAg may be involved in the pathogenesis of HBV-associated ALF. 2. Molecular mechanisms of pathogenesis of chronic liver diseases Cirrhosis develops in 30-40% of patients with chronic hepatitis B and C, but up to 80% in those with chronic hepatitis D. It may be a stable disease for decades or it may rapidly lead to liver-related death for decompensation or HCC. This variable clinical outcome suggests that not all cirrhosis is the same. To investigate these questions, we started extensive transcriptional studies in patients with end-stage liver cirrhosis of different etiology (HBV, HCV, HDV, alcohol, and autoimmune), as well as normal liver donors as a control group. Our data show a distinct gene signature for each type of cirrhosis, with a striking and unexpected heterogeneity even between biologically related conditions such as HBV and HDV cirrhosis, which may have implications for pathogenesis, treatment and natural history of these diseases. Interestingly, among liver donors, we found genes differentially expressed according to age and gender, and we are now determining whether these specific gene signatures are associated with innate or adaptive immune responses, increased fibrinogenesis, or decreased fibrinolysis since an older age (>40) at the time of infection and the male gender are associated with a worse outcome. 3. Pathogenesis of HCC and search for biomarkers for the early detection of HCC Hepatitis viruses (HBV, HCV and HDV) account for about 80% of HCC, and cirrhosis is the single most important risk factor. However, the molecular mechanisms of hepatocarcinogenesis are still unclear. Our major goal is to investigate the molecular mechanisms of hepatocarcinogenesis by studying simultaneously both the host and the virus. Because of the difficulty in studying sequential liver samples from the same patient progressing toward HCC, we investigated by microarray the molecular heterogeneity within and outside the tumor by mapping the entire liver containing HCC (up to 21 specimens for each patient). We have so far analyzed by microarray 462 liver specimens from patients with HCC of different etiology. Selected samples from the tumor and the periphery were also analyzed after laser microdissection. Remarkably, we found a high proportion of differentially expressed genes that were confirmed using both whole and microdissected tissue, confirming the robustness of our data. Interestingly, each tumor showed a distinct molecular signature with pathways specific for each tumor. An enrichment of genes related to the Ras oncogene family was documented in HBV-associated HCC and confirmed by Pathway Analysis (IPA9), whereas ATM signaling and methionine metabolism were the top-scored network functions in HCV-associated HCC. There are currently no data on the molecular pathogenesis of cirrhosis and HCC associated with HDV, a unique defective RNA virus that uses the host RNA polymerase II for replication. Our data provide the first evidence that genetic instability is the most important feature of HDV-associated HCC. We are now comparing the gene expression profiling of cirrhosis without HCC versus cirrhosis containing the tumor to see whether genes associated with liver cirrhosis are involved in the progression of cirrhosis toward HCC. The results of these studies may pave the way for the identification of new markers to predict the development of HCC, which are badly needed for the early diagnosis of HCC. 4. Correlation between viral evolution and clinical outcome in HCV infection The early evolution of the HCV quasispecies was shown to predict the outcome of acute hepatitis C (Farci et. al Science 2000), but little is known on whether the pattern of viral evolution in progressing hepatitis differs between slow and rapid progressors. Six patients were selected to represent two different clinical outcomes: 3 were slow progressors with a stable disease for >20 yrs and 3 had a rapidly progressive disease leading to liver-related death within 5 to 10 years from the onset of the infection. HCV quasispecies was studied from the first PCR-positive sample, within 2 weeks of infection, for up to 23 years. A total of 1793 sequences from the E1 and E2 genes, including the hypervariable region 1, were analyzed with newly refined bioinformatic tools. Our study provides evidence for a genetic bottleneck during the early phase of chronic HCV infection, showing a correlation between persistence of pre-existing strains after the bottleneck and rapid disease progression. Thus, the effectiveness of the host immunologic control may critically affect the pace of disease progression.